Particle Manipulation System with Out-of-plane Channel and Focusing Element

ABSTRACT

A particle manipulation system uses a MEMS-based, microfabricated particle manipulation device which has an inlet channel, output channels, and a movable member formed on a substrate. The movable member moves parallel to the fabrication plane, as does fluid flowing in the inlet channel. The movable member separates a target particle from the rest of the particles, diverting it into an output channel. However, at least one output channel is not parallel to the fabrication plane. The device may be used to separate a target particle from non-target material in a sample stream. The target particle may be, for example, a stem cell, zygote, a cancer cell, a T-cell, a component of blood, bacteria or DNA sample, for example. The particle manipulation system may also include a microfluidic structure which focuses the target particles in a particular portion of the inlet channel.

CROSS REFERENCE TO RELATED APPLICATIONS

Not applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not applicable.

STATEMENT REGARDING MICROFICHE APPENDIX

Not applicable.

BACKGROUND

This invention relates to a system and method for manipulating smallparticles in a microfabricated fluid channel.

Microelectromechanical systems (MEMS) are very small, often moveablestructures made on a substrate using surface or bulk lithographicprocessing techniques, such as those used to manufacture semiconductordevices. MEMS devices may be moveable actuators, sensors, valves,pistons, or switches, for example, with characteristic dimensions of afew microns to hundreds of microns. A moveable MEMS switch, for example,may be used to connect one or more input terminals to one or more outputterminals, all microfabricated on a substrate. The actuation means forthe moveable switch may be thermal, piezoelectric, electrostatic, ormagnetic, for example. MEMS devices may be fabricated on a semiconductorsubstrate which may manipulate particles passing by the MEMS device in afluid stream.

In another example, a MEMS devices may be a movable valve, used as asorting mechanism for sorting various particles from a fluid stream,such as cells from blood. The particles may be transported to thesorting device within the fluid stream enclosed in a microchannel, whichflows under pressure. Upon reaching the MEMS sorting device, the sortingdevice directs the particles of interest such as a blood stem cell, to aseparate receptacle, and directs the remainder of the fluid stream to awaste receptacle.

MEMS-based cell sorter systems may have substantial advantages overexisting fluorescence-activated cell sorting systems (FACS) known asflow cytometers. Flow cytometers are generally large and expensivesystems which sort cells based on a fluorescence signal from a tagaffixed to the cell of interest. The cells are diluted and suspended ina sheath fluid, and then separated into individual droplets via rapiddecompression through a nozzle. After ejection from a nozzle, thedroplets are separated into different bins electrostatically, based onthe fluorescence signal from the tag. Among the issues with thesesystems are cell damage or loss of functionality due to thedecompression, difficult and costly sterilization procedures betweensample, inability to re-sort sub-populations along different parameters,and substantial training necessary to own, operate and maintain theselarge, expensive pieces of equipment. For at least these reasons, use offlow cytometers has been restricted to large hospitals and laboratoriesand the technology has not been accessible to smaller entities.

A number of patents have been granted which are directed to suchMEMS-based particle sorting devices. For example, U.S. Pat. No.6,838,056 (the '056 patent) is directed to a MEMS-based cell sortingdevice, U.S. Pat. No. 7,264,972 b1 (the '972 patent) is directed to amicromechanical actuator for a MEMS-based cell sorting device. U.S. Pat.No. 7,220,594 (the '594 patent) is directed to optical structuresfabricated with a MEMS cell sorting apparatus, and U.S. Pat. No.7,229,838 (the '838 patent) is directed to an actuation mechanism foroperating a MEMS-based particle sorting system. Additionally, U.S.patent application Ser. No. 13/374,899 (the '899 application) and Ser.No. 13/374,898 (the '898 application) provide further details of otherMEMS designs. Each of these patents ('056, '972, '594 and '838) andpatent applications ('898 and '899) is hereby incorporated by reference.

SUMMARY

One feature of the MEMS-based microfabricated particle sorting system isthat the fluid may be confined to small, microfabricated channels formedin a semiconductor substrate throughout the sorting process. The MEMSdevice may be a valve which separates one or more target particles fromother components of a sample stream. The MEMS device may redirect theparticle flow from one channel into another channel, when a signalindicates that a target particle is present. This signal may be photonsfrom a fluorescent tag which is affixed to the target particles andexcited by laser illumination in an interrogation region upstream of theMEMS device. Thus, the MEMS device may be a particle or cell sorteroperating on a fluid sample confined to a microfabricated fluidicchannel, but using detection means similar to a FACS flow cytometer. Inparticular, the '898 application discloses a microfabricated fluidicvalve wherein the inlet channel, sort channel and waste channel all flowin a plane parallel to the fabrication plane of the microfabricatedfluidic valve.

A substantial improvement may be made over the prior art devices byhaving at least one of the microfabricated fluidic channels route theflow out of the plane of fabrication of the microfabricated valve. Avalve with such an architecture has the advantage that the pressureresisting the valve movement is minimized when the valve opens orcloses, because the movable member is not required to move a column offluid out of the way. Instead, the fluid containing the non-targetparticles may move over and under the movable member to reach the wastechannel. Furthermore, the force-generating apparatus may be disposedcloser to the movable valve, resulting in higher forces and fasteractuation speeds. As a result, the time required to open or close thevalve may be much shorter than the prior art valve, improving sortingspeed and accuracy. The systems and methods disclosed here may describesuch a microfabricated particle sorting device with at least oneout-of-plane channel.

In the systems and methods disclosed here, a micromechanical particlemanipulation device may be formed on a surface of a fabricationsubstrate, wherein the micromechanical particle manipulation device mayinclude a microfabricated, movable member having a first divertingsurface, wherein the movable member moves from a first position to asecond position in response to a force applied to the movable member,wherein the motion is substantially in a plane parallel to the surface,a sample inlet channel formed in the substrate and through which a fluidflows, the fluid including at least one target particle and non-targetmaterial, wherein the flow in the sample inlet channel is substantiallyparallel to the surface, and a plurality of output channels into whichthe microfabricated member diverts the fluid, and wherein the flow in atleast one of the output channels is not parallel to the plane, whereinat least one output channel is located directly below at least a portionof the microfabricated diverter over at least a portion of its motion.In one embodiment, The micromechanical particle manipulation device ofclaim 1, wherein the first diverting surface has a smoothly curved shapewhich is substantially tangent to the direction of flow in the inletchannel at one point on the shape and substantially tangent to thedirection of flow of a first output channel at a second point on theshape, wherein the first diverting surface diverts flow from the inletchannel into the first output channel when the movable member is in thefirst position, and allows the flow into a second output channel in thesecond position.

Finally, the systems and methods disclosed herein, because they includemicrofabricated channels as well as the novel valve design, may allowadditional useful features to be implemented. For example, thetechniques may form a particle manipulation system with cytometriccapability, as described in co-pending U.S. patent application Ser. No.13/507,830 (Owl-Cytometer) filed Aug. 1, 2012 and assigned to the sameassignee as the present application. This patent application isincorporated by reference in its entirety. The MEMS device describe heremay be used to manipulate the particles in the fluid stream enclosed inthe microfabricated channel, while a plurality of interrogation regionsalso exist which may provide feedback on the manipulation. For example,in the case of cell sorting, one laser interrogation region may existupstream of the MEMS device, and at least one additional laserinterrogation region may exist downstream of the MEMS device, to confirmthe results of the particle manipulation, that the correct cell has beensorted.

The systems and methods disclosed here also enable the construction of asingle-input/double output sorting device, wherein the flow from asingle input channel can be diverted into either of two sort outputchannels, or allowed to flow through to the waste channel.

In another embodiment, the novel valve architecture may make use ofhydrodynamic particle focusing techniques, as taught by, for example,“Single-layer planar on-chip flow cytometer using microfluidic driftingbased three-dimensional (3D) hydrodynamic focusing,” by Xaiole Mao, etal. (hereinafter “Mao,” Journal of Royal Society of Chemistry, Lab Chip,2009, 9, 1583-1589). The microfabricated architecture of the systems andmethods disclosed herein make them especially suitable for thetechniques disclosed in Mao, as described further below.

These and other features and advantages are described in, or areapparent from, the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Various exemplary details are described with reference to the followingfigures, wherein:

FIG. 1 is a simplified plan view of a microfabricated particle sortingsystem in the quiescent (no sort) position;

FIG. 2 is a simplified plan view of a microfabricated particle sortingsystem in the actuated (sort) position;

FIG. 3a is a simplified plan view of a microfabricated particle sortingsystem showing the field of view of the detector, with the microfluidicvalve in the quiescent (no sort) position; FIG. 3b is a simplifiedillustration of a microfabricated particle sorting system showing thefield of view of the detector, with the microfluidic valve in theactuated (sort) position;

FIG. 4a is a simplified cross sectional view of a microfabricatedparticle sorting system in the actuated (sort) position, showing theflow of the sample stream into the sort channel which is in the sameplane as the inlet channel; FIG. 4b is a simplified cross sectional viewof a microfabricated particle sorting system in the quiescent (no sort)position, showing the flow of the sample stream into the waste channelwhich is not in the same plane as the inlet channel; FIG. 4c is asimplified cross sectional view of a microfabricated particle sortingsystem in the quiescent (no sort) position, showing the flow of thesample stream into the waste channel which is not in the same plane asthe inlet channel, wherein the sample stream flows around the top andthe bottom of the diverter;

FIG. 5 is a simplified plan view of a microfabricated particle sortingsystem in the quiescent (no sort) position, showing the stationarymagnetically permeable feature;

FIG. 6 is a plan view of the actuation mechanism for the microfabricatedparticle sorting system, showing the functioning of the externalmagnetic field in combination with the stationary magnetically permeablefeature;

FIG. 7 is a plan view of the actuation mechanism for the microfabricatedparticle sorting system, showing the functioning of the externalmagnetic field in combination with the stationary magnetically permeablefeature, in the actuated (sort) position;

FIG. 8 is a simplified view of the microfabricated particle sortingsystem, wherein multiple microfabricated particle sorters are arrangedto provide a serial sorting capability;

FIG. 9 is a plan view of a two-way microfabricated particle sortingsystem, wherein the system has more than one sort output;

FIG. 10 is a plan view of the a two-way microfabricated particle sortingsystem, with more than one sort output, with the two-way microfabricatedparticle sorting device in the actuated position;

FIG. 11 is a plan view of the microfabricated particle sorting system incombination with a hydrodynamic focusing manifold;

FIG. 12 is a system-level illustration of a microfabricated particlesorting system according to the present invention, showing the placementof the various detection and control components; and

FIG. 13 is a representation of a signal waveform from the control systemto the microfabricated particle sorting device, showing the different inpulses used to control the motion of the device.

DETAILED DESCRIPTION

The system described herein is a particle sorting system which may makeuse of the microchannel architecture of a MEMS particle manipulationsystem. More generally, the systems and methods describe a particlemanipulation system with an inlet channel and a plurality of outputchannels, wherein at least one of the plurality of output channels isdisposed in a different plane than the inlet channel. This architecturehas some significant advantages relative to the prior art.

In the figures discussed below, similar reference numbers are intendedto refer to similar structures, and the structures are illustrated atvarious levels of detail to give a clear view of the important featuresof this novel device. It should be understood that these drawings do notnecessarily depict the structures to scale, and that directionaldesignations such as “top,” “bottom,” “upper,” “lower,” “left” and“right” are arbitrary, as the device may be constructed and operated inany particular orientation. In particular, it should be understood thatthe designations “sort” and “waste” are interchangeable, as they onlyrefer to different populations of particles, and which population iscalled the “target” or “sort” population is arbitrary.

FIG. 1 is an plan view illustration of the novel microfabricated fluidicdevice 10 in the quiescent (un-actuated) position. The device 10 mayinclude a microfabricated fluidic valve or movable member 110 and anumber of microfabricated fluidic channels 120, 122 and 140. The fluidicvalve 110 and microfabricated fluidic channels 120, 122 and 140 may beformed in a suitable substrate, such as a silicon substrate, using MEMSlithographic fabrication techniques as described in greater detailbelow. The fabrication substrate may have a fabrication plane in whichthe device is formed and in which the movable member 110 moves.

A sample stream may be introduced to the microfabricated fluidic valve110 by a sample inlet channel 120. The sample stream may contain amixture of particles, including at least one desired, target particleand a number of other undesired, nontarget particles. The particles maybe suspended in a fluid. For example, the target particle may be abiological material such as a stem cell, a cancer cell, a zygote, aprotein, a T-cell, a bacteria, a component of blood, a DNA fragment, forexample, suspended in a buffer fluid such as saline. The inlet channel120 may be formed in the same fabrication plane as the valve 110, suchthat the flow of the fluid is substantially in that plane. The motion ofthe valve 110 is also within this fabrication plane. The decision tosort/save or dispose/waste a given particle may be based on any numberof distinguishing signals. In one exemplary embodiment, the decision isbased on a fluorescence signal emitted by the particle, based on afluorescent tag affixed to the particle and excited by an illuminatinglaser. Details as to this detection mechanism are well known in theliterature, and further discussed below with respect to FIG. 12.However, other sorts of distinguishing signals may be anticipated,including scattered light or side scattered light which may be based onthe morphology of a particle, or any number of mechanical, chemical,electric or magnetic effects that can identify a particle as beingeither a target particle, and thus sorted or saved, or an nontargetparticle and thus rejected or otherwise disposed of.

With the valve 110 in the position shown, the input stream passesunimpeded to an output orifice and channel 140 which is out of the planeof the inlet channel 120, and thus out of the fabrication plane of thedevice 10. That is, the flow is from the inlet channel 120 to the outputorifice 140, from which it flows substantially vertically, and thusorthogonally to the inlet channel 120. This output orifice 140 leads toan out-of-plane channel that may be perpendicular to the plane of thepaper showing FIG. 1, and depicted in the cross sectional views of FIGS.4a-4c . More generally, the output channel 140 is not parallel to theplane of the inlet channel 120 or sort channel 122, or the fabricationplane of the movable member 110.

The output orifice 140 may be a hole formed in the fabricationsubstrate, or in a covering substrate that is bonded to the fabricationsubstrate. A relieved area above and below the sorting valve or movablemember 110 allows fluid to flow above and below the movable member 110to output orifice 140, and shown in more detail in FIGS. 4a-4c .Further, the valve 110 may have a curved diverting surface 112 which canredirect the flow of the input stream into a sort output stream, asdescribed next with respect to FIG. 2. The contour of the orifice 140may be such that it overlaps some, but not all, of the inlet channel 120and sort channel 122. By having the contour 140 overlap the inletchannel, and with relieved areas described above, a route exists for theinput stream to flow directly into the waste orifice 140 when themovable member or valve 110 is in the un-actuated waste position.

FIG. 2 is a plan view of the microfabricated device 10 in the actuatedposition. In this position, the movable member or valve 110 is deflectedupward into the position shown in FIG. 2. The diverting surface 112 is asorting contour which redirects the flow of the inlet channel 120 intothe sort output channel 122. The output channel 122 may lie insubstantially the same plane as the inlet channel 120, such that theflow within the sort channel 122 is also in substantially the same planeas the flow within the inlet channel 120. There may be an angle αbetween the inlet channel 120 and the sort channel 122, This angle maybe any value up to about 90 degrees. Actuation of movable member 110 mayarise from a force from force-generating apparatus 400, showngenerically in FIG. 2. In some embodiments, force-generating apparatusmay be an electromagnet, however, it should be understood thatforce-generating apparatus may also be electrostatic, piezoelectric, orsome other means to exert a force on movable member 110, causing it tomove from a first position (FIG. 1) to a second position (FIG. 2).

More generally, the micromechanical particle manipulation device shownin FIGS. 1 and 2 may be formed on a surface of a fabrication substrate,wherein the micromechanical particle manipulation device may include amicrofabricated, movable member 110 having a first diverting surface112, wherein the movable member 110 moves from a first position to asecond position in response to a force applied to the movable member,wherein the motion is substantially in a plane parallel to the surface,a sample inlet channel 120 formed in the substrate and through which afluid flows, the fluid including one or more target particles andnon-target material, wherein the flow in the sample inlet channel issubstantially parallel to the surface, and a plurality of outputchannels 122, 140 into which the microfabricated member diverts thefluid, and wherein the flow in at least one of the output channels 140is not parallel to the plane, and wherein at least one output channel140 is located directly below at least a portion of the movable member110 over at least a portion of its motion.

In one embodiment, the diverting surface 112 may be nearly tangent tothe input flow direction as well as the sort output flow direction, andthe slope may vary smoothly between these tangent lines. In thisembodiment, the moving mass of the stream has a momentum which issmoothly shifted from the input direction to the output direction, andthus if the target particles are biological cells, a minimum of force isdelivered to the particles. As shown in FIGS. 1 and 2, themicromechanical particle manipulation device 10 has a first divertingsurface 112 with a smoothly curved shape, wherein the surface which issubstantially tangent to the direction of flow in the sample inletchannel at one point on the shape and substantially tangent to thedirection of flow of a first output channel at a second point on theshape, wherein the first diverting surface diverts flow from the sampleinlet channel into the first output channel when the movable member 110is in the first position, and allows the flow into a second outputchannel in the second position.

In other embodiments, the overall shape of the diverter 112 may becircular, triangular, trapezoidal, parabolic, or v-shaped for example,but the diverter serves in all cases to direct the flow from the inletchannel to another channel.

It should be understood that although channel 122 is referred to as the“sort channel” and orifice 140 is referred to as the “waste orifice”,these terms can be interchanged such that the sort stream is directedinto the waste orifice 140 and the waste stream is directed into channel122, without any loss of generality. Similarly, the “inlet channel” 120and “sort channel” 122 may be reversed. The terms used to designate thethree channels are arbitrary, but the inlet stream may be diverted bythe valve 110 into either of two separate directions, at least one ofwhich does not lie in the same plane as the other two. The term“substantially” when used in reference to an angular direction, i.e.substantially tangent or substantially vertical, should be understood tomean within 15 degrees of the referenced direction. For example,“substantially orthogonal” to a line should be understood to mean fromabout 75 degrees to about 105 degrees from the line.

FIGS. 3a and 3b illustrate an embodiment wherein the angle α between theinlet channel 120 and the sort channel 122 is approximately zerodegrees. Accordingly, the sort channel 122 is essentially antiparallelto the inlet channel 120, such that the flow is from right to left inthe inlet channel 120. With valve 110 in the un-actuated, quiescentposition shown in FIG. 3a , the inlet stream flows straight to the wasteorifice 140 and vertically out of the device 10.

In FIG. 3b , the valve 110 is in the actuated, sort position. In thisposition, the flow is turned around by the diverting surface 112 of thevalve 110 and into the antiparallel sort channel 122. This configurationmay have an advantage in that the field of view of the detector 150covers both the inlet channel 120 and the sort channel 122. Thus asingle set of detection optics may be used to detect the passage of atarget particle through the respective channels. It may also beadvantageous to minimize the distance between the detection region andthe valve 110, in order to minimize the timing uncertainty in theopening and closing of the valve.

The movable member or valve 110 may be attached to the substrate with aflexible spring 114. The spring may be a narrow isthmus of substratematerial. In the example set forth above, the substrate material may besingle crystal silicon, which is known for its outstanding mechanicalproperties, such as its strength, low residual stress and resistance tocreep. With proper doping, the material can also be made to besufficiently conductive so as to avoid charge build up on any portion ofthe device, which might otherwise interfere with its movement. Thespring may have a serpentine shape as shown, having a width of about 1micron to about 10 microns and a spring constant of between about 10 N/mand 100 N/m, and preferably about 40 N/m

FIGS. 4a, 4b, 4c are cross sectional views illustrating the operation ofthe out-of-plane waste channel 140. FIG. 4c is slightly enlargedrelative to FIGS. 4a and 4b , in order to show detail of the flow aroundthe movable member 110 and into the waste channel 142 through wasteorifice 140. In this embodiment, the waste channel 142 is vertical,substantially orthogonal to the inlet stream 120 and sort stream 122. Itshould be understood that other embodiments are possible other thanorthogonal, but in any event, the flow into waste channel 142 is out ofthe plane of the flow in the inlet channel 120 and/or sort channel 122.As shown in FIG. 4a , with the valve in the sort, actuated position, theinlet stream and target particle may flow into the sort stream, which inFIG. 4a is out of the paper, and the waste orifice 140 is largely,though not completely, blocked by the movable member 110. The area 144(shown more clearly in FIG. 4c ) on top of the valve or movable member110 may be relieved to provide clearance for this flow.

When the valve or movable member 110 is un-actuated as in FIG. 4b , theflow of the inlet channel 120 may flow directly into the waste channel142 by going over, around or by the movable member or valve 110. Thearea 144 on top of the valve or movable member 110 may be relieved toprovide clearance for this flow. The relieved area 144 is shown ingreater detail in the enlarged FIG. 4c . Thus when the movable member isun-actuated, the flow will be sent directly to the waste channel. Whenthe movable member is actuated, most of the fluid will be directed tothe sort channel, although liquid may still flow over and under themovable member.

Thus, the purpose of providing flow both under and over the movablemember 110 is to reduce the fluid pressure produced by the actuatormotion in the region behind the valve or movable member 110. In otherwords, the purpose is to provide as short a path as possible between thehigh pressure region in front of the valve 110 and the low pressureregion behind the valve. This allows the valve to operate with littlepressure resisting its motion. As a result, the movable valve 110 shownin FIGS. 1-4 c may be substantially faster than valves which have allchannels disposed in the same plane.

Another advantage of the vertical waste channel 142 is that bypositioning it directly underneath a stationary permeable feature 130and movable permeable feature 116, the magnetic gap between thepermeable features 116 and 130 can be narrower than if the fluidicchannel went between them. The narrower gap enables higher forces andthus faster actuation compared to prior art designs. A description ofthe magnetic components and the magnetic actuation mechanism will begiven next, and the advantages of the out-of-plane channel architecturewill be apparent.

FIG. 5 is a plan view of another exemplary embodiment of device 100 ofthe device 10, showing the disposition of a stationary permeable feature130 and further detail of the movable member 110. In this embodiment,the movable member 110 may include the diverting surface 112, theflexible hinge or spring 114, and a separate area 116 circumscribed butinside the line corresponding to movable member 110. This area 116 maybe inlaid with a permeable magnetic material such as nickel-ironpermalloy, and may function as described further below.

A magnetically permeable material should be understood to mean anymaterial which is capable of supporting the formation of a magneticfield within itself. In other words, the permeability of a material isthe degree of magnetization that the material obtains in response to anapplied magnetic field.

The terms “permeable material” or “material with high magneticpermeability” as used herein should be understood to be a material witha permeability which is large compared to the permeability of air orvacuum. That is, a permeable material or material with high magneticpermeability is a material with a relative permeability (compared to airor vacuum) of at least about 100, that is, 100 times the permeability ofair or vacuum which is about 1.26×10⁻⁶ H·m⁻¹. There are many examples ofpermeable materials, including chromium (Cr), cobalt (Co), nickel (Ni)and iron (Fe) alloys. One popular permeable material is known asPermalloy, which has a composition of between about 60% and about 90% Niand 40% and 10% iron. The most common composition is 80% Ni and 20% Fe,which has a relative permeability of about 8,000.

It is well known from magnetostatics that permeable materials are drawninto areas wherein the lines of magnetic flux are concentrated, in orderto lower the reluctance of the path provided by the permeable materialto the flux. Accordingly, a gradient in the magnetic field urges themotion of the movable member 110 because of the presence of inlaidpermeable material 116, towards areas having a high concentration ofmagnetic flux. That is, the movable member 110 with inlaid permeablematerial 116 will be drawn in the direction of positive gradient inmagnetic flux.

An external source of magnetic field lines of flux may be providedoutside the device 100, as shown in FIG. 6. This source may be anelectromagnet 500. The electromagnet 500 may include a permeable core512 around which a conductor 514 is wound. The wound conductor or coil514 and core 512 generate a magnetic field which exits the pole of themagnet, diverges, and returns to the opposite pole, as is well knownfrom electromagnetism. Accordingly, the movable member 110 is generallydrawn toward the pole of the electromagnet 500 as shown in FIG. 7.

However, the performance of the device 100 can be improved by the use ofa stationary permeable feature 130. The term “stationary feature” shouldbe understood to mean a feature which is affixed to the substrate anddoes not move relative to the substrate, unlike movable member or valve110. A stationary permeable feature 130 may be shaped to collect thesediverging lines of flux and refocus them in an area directly adjacent tothe movable member 110 with inlaid permeable material. The stationarypermeable feature may have an expansive region 132 with a narrowerthroat 134. The lines of flux are collected in the expansive region 132and focused into and out of the narrow throat area 134. Accordingly, thedensity of flux lines in the throat area 134 is substantially higherthan it would be in the absence of the stationary permeable feature 130.Thus, use of the stationary permeable feature 130 though optional,allows a higher force, faster actuation, and reduces the need for theelectromagnet 500 to be in close proximity to the device 10. From thenarrow throat area 134, the field lines exit the permeable material andreturn to the opposite magnetic pole of the external source 500. Butbecause of the high concentration of field lines in throat area 134, thepermeable material 116 inlaid into movable member 110 may be drawntoward the stationary permeable feature 130, bringing the rest ofmovable member with it.

When the electromagnet is quiescent, and no current is being supplied tocoil 514, the restoring force of spring 114 causes the movable member110 to be in the “closed” or “waste” position. In this position, theinlet stream passes unimpeded through the device 100 to the wastechannel 140. This position is shown in FIG. 5. When the electromagnet500 is activated, and a current is applied through coil 514, a magneticfield arises in the core 512 and exits the pole of the core 512. Theselines of flux are collected and focused by the stationary permeablefeature 130 and focused in the region directly adjacent to the throat134. As mentioned previously, the permeable portion 116 of the movablemember 110 is drawn toward the throat 134, thus moving the movablemember 110 and diverting surface 112 such that the inlet stream in inletchannel 120 is redirected to the output or sort channel 122. Thisposition is shown in FIG. 7.

Permalloy may be used to create the permeable features 116 and 130,although it should be understood that other permeable materials may alsobe used. Permalloy is a well known material that lends itself to MEMSlithographic fabrication techniques. A method for making the permeablefeatures 116 and 130 is described further below.

As mentioned previously, having the waste channel 140 and 142 directlybeneath the movable member or valve 110 allows the movable permeablefeature 116 to be disposed much closer to the stationary permeablefeature 130. If instead the waste channel were in the same plane, thisgap would have to be at least large enough to accommodate the wastechannel, along with associated tolerances. As a result, actuation forcesare higher and valve opening and closing times are much shorter. This inturn corresponds to either faster sorting or better sorting accuracy, orboth.

With the use of the electromagnetic actuation technique described above,actuation times on the order of 10 microseconds can be realized.Accordingly, the particle sorting device is capable of sorting particlesat rates in excess of 50 kHz or higher, assuming 10 microsecondsrequired to pull the actuator in, and 10 microseconds required to returnit to the as-manufactured position.

For any particle sorting mechanism however, there is an inherenttrade-off between sort purity and sort speed. One can only increase thefluid speed to a certain point, after which one runs into physicallimitations of the sorter, for example, when the valve speed is suchthat there is insufficient time to open the valve or flap when a cell isdetected. Beyond that limitation, the most obvious way to achieve moreevents per second is to increase the cell density. But, with increasedcell density, the incidence of sort conflicts, wherein both a desiredand an undesired cell are collected, also increases.

In order to overcome this limitation, a cell sample may theoretically beprocessed multiple times in a sequential sort strategy—initially a veryrapid, crude sort followed by a—slower, high precision sort. This isgenerally not a practical option with a traditional FACS system as aresult of massive cell dilution (from sheath fluid), slow processingspeeds and unacceptable cell damage resulting from multiple passesthrough the high pressure electrostatic sorting mechanism. A single passthrough a flow cytometer is exceptionally violent, with 10 msecvelocities, explosive decompression from 60 psi to 0 psi. Cells areunlikely to survive such treatment on multiple passes withoutsignificant loss of viability. Even if one is willing to accept thedilution, manual processing and cell death, the yield losses on a FACSwould be overwhelming. Also, the time constant per cycle for processing,cleaning, sterilization and certification is untenable and the sterilityof the sample is completely compromised. As a result, this sequentialsorting is not a practical approach for FACS-based clinical cellsorting.

In contrast, for the microfabricated particle sorting system describedabove, using the microfluidic channel architecture, a multi-stage,“sequential” sort may be performed in a straightforward way as describedbelow. A plurality of particle manipulation operations may take placeusing a plurality of MEMS sorting devices 10 or 100. The sorting devicesmay be on separate MEMS chips and enclosed in a disposable cartridge, ormultiple valves may be formed on a single substrate using MEMSfabrication techniques. In one embodiment, the plurality of MEMS sortingchips are separated by some extent, such that by laterally shifting thedevice, the additional MEMS chips may become operational. Thisembodiment is described further below, and illustrated in FIG. 8. Morebroadly, the sorting device system may include a secondary manipulationdevice or sorting stage 200 downstream of the first manipulation deviceor sorting stage 100. Sorting stage 100 connotes a stage using eitherdevice 10 or device 100 for example, as illustrated in FIGS. 1 and 5,respectively.

The first sorting stage 100 and second sorting stage 200 are bothpreceded by a laser interrogation region 170 and 270, respectively. Inthis region, a laser is used to irradiate the particles in the samplestream. Those particles bearing a fluorescent tag may fluoresce as aresult of the laser irradiation. This fluorescence signal is detectedand is indicative of the presence of a target particle in the samplestream. Upon detection of the target particle, a signal is sent to thecontroller controlling the electromagnet 500, energizing theelectromagnet and thus opening the movable member or valve 110. Thetarget particle is thus directed into the sort channel 122. Thisfunctionality is described in further detail below with respect to thefull particle sorting system shown in FIG. 12. The sorting stages 100and 200 may also be accompanied by a third laser interrogation region280 downstream of the last sorting stage 200. This interrogation may beperformed to evaluate the accuracy of the sort, or in order to adjustvarious sorting parameters. Although only two sorting operationsarranged sequentially are shown in FIG. 12, it should be understood thatthis basic concept may be extended to any number of additional sortingstages, and that the stages may be arranged in a parallel configuration,instead of, or in addition to, the serial configuration.

Accordingly, a first sort may be run rapidly through a first sortingstage 100, to enrich target cells with negligible yield losses. Theoutput of the first sorting stage 100 may flow into either a wastechannel 140 or a sort channel 122, based on the output of adiscriminator or detector located in region 170. If the stream flows tothe sort channel 122, it then flows on to a second sorting stage 200,which may have its own associated detection area 270. Similarly to sortstage 100, the flow may be direct to a waste channel 240 or a sortchannel 222. Using this approach, the sample remains sterile and gentlyhandled through the entire sequential sorting process. It should beunderstood that although difficult to depict in a two dimensionaldrawing, the waste channel 140 and 240 may lie in a different planerelative to the inlet channel 120, and sort channels 122 and 222. InFIG. 8 waste channels 140 and 240 are depicted flowing into the paper.

In another embodiment, using the architecture shown in FIG. 1, 3, or 5,a dual output, dual position particle manipulation device may also beenvisioned. Such a device is shown in FIG. 9. FIG. 9 shows a dual outputdevice 800 wherein a single inlet channel 820 can feed either of twoseparate sort output channels 822 and 824, depending on the position ofmovable member 810. Dual output device 800 may have two permeable areas816 and 818, which may be drawn toward either of two stationarypermeable features 830 and 850, respectively. For example, if a sourceof external magnetic flux such as electromagnet 500 is positioned nearstationary permeable feature 830, the flux emitted from electromagnet500 is concentrated by stationary permeable feature 830 and movablepermeable feature 816 is drawn toward it. The situation is as depictedin FIG. 10. When the movable feature rotates clockwise, opening sortchannel 822 to the flow from inlet channel 820 by diverting surface 842.When another external magnet (not shown) is energized above device 800and upper stationary permeable feature 850, the movable member 810rotates counterclockwise, directing the flow in inlet channel 820 intothe upper sort channel 824 by sort diverting surface 812. The wastechannel orifice 840 may be enlarged compared to 140, such that it isdisposed directly under at least a portion of movable member 810, butdoes not interfere with the motion of sort diverting surfaces 812 or842.

Although the embodiments shown in FIGS. 1-11 are described with respectto an electromagnetic actuation mechanism, it should be understood thatother actuation forces may be used instead. For example, if permeablefeatures 116 and 130 are made from an electrically conductive ratherthan permeable magnetic material, a voltage potential may be placedacross elements 116 and 130, producing an electrostatic force to movethe movable member 110. Piezoelectric forces may also be used.

Because of the microfabricated architecture of particle manipulationdevice 10 and 100, it lends itself to techniques that can make use ofsuch an enclosed, well defined architecture. One such technique isillustrated in FIG. 11, wherein the microfabricated particlemanipulation device may have at least one additional channel thatprovides a sheath fluid to the sample stream and also a focusing elementcoupled to the inlet channel. The sheath fluid may be used to adjust theconcentration or positioning of the target particles within the inletchannel. The focusing element may be configured to urge the targetparticles into a particular portion of the sample inlet channel, asdescribed further below. The focusing element may be disposed insubstantially the same plane as the movable member 110, and may beformed in the same substrate surface as the movable member 110 and inletchannel 120.

FIG. 11 depicts a microfabricated fluidic manifold 300 which may be usedto focus the particles in a certain area within the fluid stream.Techniques for designing such a manifold may be found in, for example,“Single-layer planar on-chip flow cytometer using microfluidic driftingbased three-dimensional (3D) hydrodynamic focusing,” by Xiaole Mao etcl, Journal of Royal Society of Chemistry, Lab Chip, 2009, 9, 1583-1589.The manifold may include a sample inlet 310 and sheath fluid channel320. As the name suggests, the sheath channel adds a sheath fluid to thesample stream, which is a buffering fluid which tends to dilute the flowof particles in the stream and locate them in a particular portion ofthe stream. The combined fluid then flows around a focusing elementcoupled to the inlet channel 120, here a z-focusing channel 330, whichtends to herd the particles into a particular plane within the flow.This plane is substantially in the plane of the paper of FIG. 11. Thecombined fluid then passes another intersection point, a “y-intersectionpoint” 350, which introduces additional sheath fluid above and below theplane of particles. At the y-intersection point 350, two flows may jointhe z-focus channel 330 from substantially antiparallel directions, andorthogonal to the z-focus channel 330. This intersection may compressthe plane of particles into a single point, substantially in the centerof the stream. Accordingly, at the y-intersection point 350 the targetparticles may be compressed from a plane to a stream line near thecenter of the z-focus channel 330 and sample inlet channel 120. Focusingthe particles into a certain volume tends to decrease the uncertainly intheir location, and thus the uncertainty in the timing of the openingand closing of the movable member or valve 110. Such hydrodynamicfocusing may therefore improve the speed and/or accuracy of the sortingoperation.

In one exemplary embodiment of the microfabricated particle manipulationdevice 10 or 100 with hydrodynamic focusing illustrated in FIG. 11, theangular sweep of z-bend 330 is a curved arc of about 180 degrees. Thatis, the approximate angular sweep between the junction of the sheathinlet with the cell inlet and the y-intersection point 350, may be about180 degrees. Generally, the radius of curvature of the z-bend 330 may beat least about 100 microns and less than about 500 microns, and thecharacteristic dimension, that is the width, of the channels istypically about 50 microns to provide the focusing effect. In oneembodiment, the radius of curvature of the channel may be about 250microns, and the channel widths, or characteristic dimensions, for thesample inlet channel 120 and z-bend channel are on the order of about 50microns. These characteristic dimensions may provide a curvaturesufficient to focus the particles, such that they tend to be confined tothe plane of the paper upon exit from the z-focus channel 330 aty-intersection point 350. This plane is then compressed to a point inthe channel at the y-intersection point 350.

The microfabricated particle manipulation device 10 or 100 may be usedin a particle sorting system 1000 enclosed in a housing containing thecomponents shown in FIG. 12. The MEMS particle manipulation devices 10,100 or 800 may be enclosed in a plastic, disposable cartridge which isinserted into the system 1000. The insertion area may be a movable stagewith mechanisms available for fine positioning of the particlemanipulation device 10, 100 or 800 and associated microfluidic channelsagainst one or more data, which orient and position the detection regionand particle manipulation device 10, 100 or 800 with respect to thecollection optics 1100. If finer positioning is required, the inletstage may also be a translation stage, which adjusts the positioningbased on observation of the location of the movable member 110 relativeto a datum.

It should be understood that although FIG. 12 shows a particle sortingsystem 1000 which uses a plurality of laser sources 1400 and 1410, onlya single laser may be required depending on the application. For theplurality of lasers shown in FIG. 12, one of the laser sources 1410 maybe used with an associated set of parallel optics (not shown in FIG. 12)to illuminate the at least one additional laser interrogation region 170and/or 270. This setup may be somewhat more complicated and expensive toarrange than a single laser system, but may have advantages in that theoptical and detection paths may be separated for the different laserinterrogation regions. For this embodiment, it may not be necessary toalter the trajectory, spectral content, timing or duration of the laser1410 light. Although not shown explicitly in FIG. 12, it should beunderstood that the detection path for additional laser(s) 1410 may alsobe separate from the detection path for laser 1400. Accordingly, someembodiments of the particle sorting system may include a plurality oflaser sources and a plurality of optical detection paths, whereas otherembodiments may only use a single laser source 1400 and collectionoptics 1100. In the embodiment described here, a plurality of excitationlasers uses a common optical path, and the optical signals are separatedelectronically by the system shown in FIG. 12.

The embodiment shown in FIG. 12 is based on a FACS-type detectionmechanism, wherein one or more lasers 1400, 1410 excites one or morefluorescent tags affixed to the target particles. The laser excitationmay take place in multiple interrogation regions, such as regions 170,270 and 280. The fluorescence emitted as a result are detected and thesignal is fed to a computer 1900. The computer then generates a controlsignal that controls the electromagnet 500, or multiple electromagnetsif multiple sorters are used such as in FIG. 8. It should be understoodthat other detection mechanisms may be used instead, includingelectrical, mechanical, chemical, or other effects that can distinguishtarget particles from non-target particles.

Accordingly, the MEMS particle sorting system 1000 shown in FIG. 12 mayinclude a number of elements that may be helpful in implementing theadditional interrogation regions 170 and 270, or more. First, an opticalmanipulating means 1600 may alter the trajectory, spectral content,timing or duration of the laser radiation from laser 1400 to the secondor third interrogation spots. Examples of items that may be included inoptical manipulating means 1600 are a birefringent crystal, spinningprism, mirror, saturable absorber, acousto-optic modulator, harmoniccrystal, Q-switch, for example. More generally, optical manipulatingmeans 1600 may include one or more items that alter laser frequency,amplitude, timing or trajectory along one branch of the optical path toan additional interrogation region.

For example, optical manipulating means 1600 may include a beamsplitterand/or acousto-optic modulator. The beam splitter may separate a portionof the incoming laser beam into a secondary branch or arm, where thissecondary branch or arm passes through the modulator which modulates theamplitude of the secondary beam at a high frequency. The modulationfrequency may be, for example, about 2 MHz or higher. The lightimpinging on the first laser interrogation region 101 may, in contrast,be continuous wave (unmodulated). The secondary branch or arm is thendirected to the additional laser interrogation region 170 or 270. Thisexcitation will then produce a corresponding fluorescent pattern from anappropriately tagged cell.

This modulated fluorescent pattern may then be picked up by thedetection optics 1600, which may recombine the detected fluorescencefrom interrogation region 170 and/or 270 with fluorescence from laserinterrogation region 170. The combined radiation may then impinge on theone or more detectors 1300.

An additional optical component 1700 may also alter the frequency,amplitude, timing or trajectory of the second beam path, however, it mayperform this operation upstream (on the detector side) of the collectionoptics 1100 rather than downstream (on the sample side) of it, as doesoptical component 1600.

The output of detectors 1300 may be analyzed to separate the contentcorresponding to laser interrogation region 280 from the contentcorresponding to laser interrogation region 170 or 270. This may beaccomplished by applying some electronic distinguishing means to thesignals from detectors 1300. The details of electronic distinguishingmeans 1800 may depend on the choice for optical manipulation means 1600.For example, the distinguishing means 1800 may include a high pass stageand a low pass stage that is consistent with a photoacoustic modulatorthat was included in optical manipulating means 1600. Or electronicdistinguishing means 1800 may include a filter (high pass and/or lowpass) and/or an envelope detector, for example.

Therefore, depending on the choice of optical manipulating means 1600,the unfiltered signal output from detectors 1300 may include acontinuous wave, low frequency portion and a modulated, high frequencyportion. After filtering through the high pass filter stage, the signalmay have substantially only the high frequency portion, and after thelow pass stage, only the low frequency portion. These signals may thenbe easily separated in the logic circuits of computer 1900.Alternatively, the high pass filter may be an envelope detector, whichputs out a signal corresponding to the envelop of the amplitudes of thehigh frequency pulses.

Other sorts of components may be included in electronic distinguishingmeans 1800 to separate the signals. These components may include, forexample, a signal filter, mixer, phase locked loop, multiplexer,trigger, or any other similar device that can separate or distinguishthe signals. Component 1800 may also include the high pass and/or lowpass electronic filter or the envelope detector described previously.The two sets of signals from the electronic distinguishing means 1800may be handled differently by the logic circuits 1900 in order toseparate the signals.

Thus, a MEMS particle manipulation system may be used in conjunctionwith one or more additional downstream laser interrogation regions,wherein the additional laser interrogation regions are used to confirmthe effectiveness or accuracy of a manipulation stage in manipulating astream of particles. The downstream evaluation from laser interrogationregion 280 past the sorting stage 100 and 200 may allow the operator tomeasure one event number (e.g. the captured event rate post-sort)divided by another event number (e.g. the initial event rate pre-sort)for individual particle types, and to feedback to adjust initialinterrogation parameters (e.g. such as x, y, z position and also “openwindow” length in time) based on this ratio. This method may be used tooptimize the yield or accuracy of the system 1000. Alternatively, theoperator could measure the event rate post-sort of target cells, dividedby total event rate post-sort feedback to adjust initial laserinterrogation parameters such as x, y, z position and also “open window”length in time, in order to optimize the purity of the sorting system1000. These sorting parameters may be adjusted by changing controlsignal 2000 which is sent by computer 1900 to electromagnet 500, or bychanging the optical detection parameters or by changing the lasercontrol signals, as shown in FIG. 12.

One example of how the system depicted in FIG. 12 may be used to adjustthe sorting parameters, is via the control signal waveform 2000delivered to the electromagnet 500. This waveform 2000 may be fine-tunedto adjust the sorting performance of the valve or movable member 110 or810, and may be produced by logic circuits 1900. FIG. 13 depicts acontrol signal waveform 2000 with additional features that may be usedto control the motion of movable member 110 or 810. This control signalwaveform 2000 may be generated by computer 1900, and thus may be madeessentially arbitrarily complex. The control signal waveform 2000 may beeither a voltage waveform or a current waveform. The control signalwaveform 2000 may be applied to coil 510 of electromagnet 500, forexample, to drive current through the coil to produce the actuatingmagnetic field. The control signal 2000 may include an accelerationphase 2110 which has a substantially larger magnitude than the remainderof the control signal waveform 2000, and lasts for tens of microseconds.

The larger magnitude of the current in the acceleration phase may beused to overcome the back electromotive force produced in the coils bythe moving magnets. It may also produce a higher force, which may beneeded to break the movable member 110, 810 from its rest position andovercome any stiction forces that may be hindering motion. After thisinitial acceleration phase, the control signal may have a maintenancephase during which the current is essentially constant and lasts fortens of microseconds. During this period, the movable member 110 or 810travels from its closed position in FIG. 1, 5 or 9 to actuated positionsshown in FIG. 2, 7 or 10. Although the current may be constant duringthis period, the force on the movable member may be variable, a functionof the closing distance between movable permeable feature 116, 816 and840 and the respective stationary permeable features 130, 840 and 850.Reversing the polarity of the control signal as shown in 2130 reversesthe direction of the magnetic field, and demagnetizes the permeableportions. After the reversal period 2130, a quiescent period 2140lasting several microseconds may follow, during which there is nomagnetic field produced, and the spring force of spring element 114 or814 on movable member 110 or 810 may return the movable member to itsun-actuated state. This may be in the waste or reject position. After aperiod when the actuator is closing and about to reach theas-manufactured position, a short “braking” pulse 2150 may slow thevelocity of the movable member. This may avoid an undesirable bounce offthe hard stop, which may otherwise allow a non-target particle to enterthe sort channel 122. Or if there is no hard stop, this may allow thefastest return to the un-actuated position.

Using the downstream confirmation of the sort channel contents asdescribed above with respect to FIG. 12, any of the adjustableparameters of the current profile shown in FIG. 13, such as amplitudeand duration of the acceleration phase, amplitude and duration of theopening phase, duration of the quiescent phase, or amplitude andduration of the braking phase, may be adjusted to improve the sortperformance of the system.

The description now turns to the fabrication of the devices shown inFIGS. 1-11. Fabrication may begin with the inlaid permeable features 116and 130 formed in a first substrate. The substrate may be a singlecrystal silicon substrate, for example. To form these structures,depressions may be formed in these areas of the substrate surface byetching. First, photoresist may be deposited over the substrate surfaceand removed over the areas corresponding to 116 and 130. Then, thetrenches may be formed by, for example, etching the substrate inpotassium hydroxide (KOH) to form a suitable depression. A seed layermay be deposited conformally over the first substrate surface andpatterned to provide the seed layer for plating NiFe into the trenches.The seed layer may be, for example, Ti/W or Cr/Au may then be depositedby sputtering, CVD or plasma deposition. This layer may be covered withphotoresist and patterned according to the desired shape of the areas116 and 130. Unwanted areas of photoresist and seed layer may then beremoved by chemical etching. The permeable features may then bedeposited over the patterned seed layer by sputtering, plasma depositionor electrochemical plating. It is known that permalloy (80% Ni and 20%Fe), for example, can readily be deposited by electroplating.

Alternatively, a liftoff method may be used to deposit a sheet ofpermeable material, most of which is then lifted off areas other than116 and 130. Further details into the lithographic formation of inlaid,magnetically permeable materials may be found in, for example, U.S. Pat.No. 7,229,838. U.S. Pat. No. 7,229,838 is hereby incorporated byreference in its entirety. The substrate may then be planarized bychemical mechanical polishing (CMP), leaving a flat surface for thelater bonding of a cover plate.

Having made the permeable features 116 and 130, the movable member orvalve 110 and 810 may be formed. The surface may again be covered withphotoresist and patterned to protect the inlaid permeable features 116and 130. The inlet channel 120 and output channels 122 and relieved area144 may be formed simultaneously with the movable member 110 and 810.With movable member 110, 810 and other areas whose topography is to bepreserved covered with photoresist, the features 110, 810, 120, 122 and144 may be formed by deep reactive ion etching (DRIE) for example.

To form the fluidic channels, a cover plate may be bonded to the surfaceof the substrate which was previously planarized for this purpose. Thecover plate may be optically transparent to allow laser light to beapplied to the particles in the fluid stream flowing in the inletchannel 120, and for fluorescence emitted by the fluorescent tagsaffixed to the particles to be detected by the optical detection systemdescribed above. A hole formed in this transparent material may form thewaste channel 142. Alternatively, a waste channel 142 may be formed in asecond substrate, such as a second silicon substrate, and bonded to thesurface of the first substrate. Alternatively, output channel 142 may beformed on the opposite surface of the first substrate using asilicon-on-insulator (SOI) substrate, with waste channel 142 and orifice140 formed in the handle layer and dielectric layer of the SOIsubstrate, and the movable feature formed in the device layer.

Additional details for carrying out this process outlined above are wellknown to those skilled in the art, or readily found in numerouslithographic processing references.

While various details have been described in conjunction with theexemplary implementations outlined above, various alternatives,modifications, variations, improvements, and/or substantial equivalents,whether known or that are or may be presently unforeseen, may becomeapparent upon reviewing the foregoing disclosure. Accordingly, theexemplary implementations set forth above, are intended to beillustrative, not limiting.

1. A micromechanical particle manipulation device, formed on a surfaceof a substrate, comprising: a microfabricated, movable member formed ona surface of the substrate in a first plane, and having a firstdiverting surface, wherein the movable member moves from a firstposition to a second position in response to a force applied to themovable member, wherein the motion is substantially in the first planeparallel to the surface of the substrate; a sample inlet channel formedin the substrate and through which a fluid flows, the fluid includingtarget particles and non-target material, wherein the flow in the sampleinlet channel is substantially in the first plane parallel to thesurface of the substrate; a sort output channel into which themicrofabricated member diverts the target particles and a waste outputchannel into which the non-target material flows, and wherein the wasteoutput channel is in a second, different plane than the movable memberand the sample inlet channel, and wherein the waste output channel islocated directly below at least a portion of the microfabricated memberover at least a portion of its motion; and a sheath fluid inlet in fluidcommunication with the sample channel; and a focusing element coupled tothe sheath fluid inlet, which is configured to urge the target particlesinto a particular portion of the sample channel.
 2. The micromechanicalparticle manipulation device of claim 1, wherein focusing elementcomprises a z-focus channel, wherein the z-focus channel curves in anarc of about 180 degree from the sheath fluid inlet, and urges thetarget particles into substantially a single plane.
 3. Themicromechanical particle manipulation device of claim 2, wherein thez-focus channel has a radius of curvature of at least about 100 micronsand less than about 500 microns.
 4. The micromechanical particlemanipulation device of claim 1, wherein the focusing element is disposedin the same plane as the movable member, and formed in the samesubstrate.
 5. The micromechanical particle manipulation device of claim2, wherein the inlet channel and z-focus channel both havecharacteristic dimensions of about 50 microns.
 6. The micromechanicalparticle manipulation device of claim 2, further comprising ay-intersection point at which the target particles are compressed fromthe plane to a stream line near the center of the z-focus channel. 7.The micromechanical particle manipulation device of claim 6, wherein they-intersection point occurs where two flows join the z-focus channelfrom substantially antiparallel directions, and each substantiallyorthogonal to the z-focus channel at the y-intersection point.
 8. Themicromechanical particle manipulation device of claim 1, wherein thetarget particles are at least one of a stem cell, a cancer cell, azygote, a protein, a T-cell, a bacteria, a component of blood, and a DNAfragment.
 9. The micromechanical particle manipulation device of claim1, wherein the first diverting surface has a shape which issubstantially tangent to the direction of flow in the input channel atone point on the shape and substantially tangent to the direction offlow of the first output channel at a second point on the shape, andwherein the first diverting surface diverts flow from the input channelinto a first output channel when the movable member is in the firstposition, and allows the flow into the second output channel in thesecond position.
 10. The micromechanical particle manipulation device ofclaim 1, wherein the plurality of output channels comprises a sortchannel and a waste channel, wherein flow in the sort channel issubstantially antiparallel to flow in the input channel, and whereinflow in the waste channel is substantially orthogonal to flow in theinput channel and the sort channel.
 11. The micromechanical particlemanipulation device of claim 1, further comprising: a first permeablemagnetic material inlaid in the movable member; a first stationarypermeable magnetic feature disposed on the substrate; and a first sourceof magnetic flux external to the movable member and substrate on whichthe movable member is formed.
 12. The micromechanical particlemanipulation device of claim 11, wherein the movable member moves fromthe first position to the second position when the source of magneticflux is activated.
 13. The micromechanical particle manipulation deviceof claim 1, wherein the force is at least one of magnetic,electrostatic, and piezoelectric.
 14. A particle manipulation system,comprising: the micromechanical particle sorting device of claim 1; atleast one laser directed to a laser interrogation region disposed in theinput channel; and at least one set of detection optics that detects afluorescent signal from a fluorescent tag affixed to the target particlein the fluid.
 15. The particle manipulation system of claim 14, furthercomprising: an electromagnet; and a circuit that provides a controlwaveform to the electromagnet.
 16. The particle manipulation system ofclaim 14, further comprising: at least one additional laser directed ata region in at least one of the output channels and configured toconfirm results of a particle manipulation.
 17. The particlemanipulation system of claim 15, wherein the control waveform includes ahigher amplitude acceleration phase which sets the movable member inmotion, a constant amplitude phase which opens the movable member, and abraking phase which slows the movable member at closure.
 18. A method ofmaking a microfabricated particle manipulation device on a surface of afabrication substrate, comprising: forming a movable member on thesurface of the substrate having a first diverting surface, wherein themovable member is configured to move from a first position to a secondposition in response to a force applied to the movable member, whereinthe motion is substantially in a first plane parallel to the surface;forming a sample inlet channel formed in the substrate and through whicha fluid flows, the fluid including at least one target particle andnon-target material, wherein the flow in the sample inlet channel issubstantially in the first plane parallel to the surface; forming aplurality of output channels into which the microfabricated memberdiverts the target particle, and wherein the flow in at least one of theoutput channels is in a second different plane than the movable memberand the sample inlet channel, and wherein at least one output channel islocated directly below at least a portion of the microfabricateddiverter over at least a portion of its motion forming a sheath fluidinlet in fluid communication with the sample channel; and forming afocusing element coupled to the sheath fluid inlet, which is configuredto urge the target particles into a particular portion of the samplechannel, wherein the focusing element is formed in the same substrate asthe movable member and inlet channel.
 19. The method of making themicrofabricated particle manipulation device of claim 18, furthercomprising: forming the diverting surface with a smoothly curved shapewhich is substantially tangent to the direction of flow in the inputchannel at one point on the shape and substantially tangent to thedirection of flow of a first output channel at a second point on theshape, wherein the first diverting surface is configured to divert flowfrom the input channel into the first output channel when the movablemember is in the first position, and allows the flow into a secondoutput channel in the second position; and wherein forming the focusingelement comprises forming a z-focus channel with a curved arc of about180 degrees from the sheath fluid inlet, wherein the z-focus channelurges the target particles into substantially a single plane.
 20. Themethod of making the microfabricated particle manipulation device ofclaim 19, further comprising: forming a sort channel and a wastechannel, wherein flow in the sort channel is substantially antiparallelto flow in the input channel, and wherein flow in the waste channel issubstantially orthogonal to flow in the input channel and the sortchannel, and wherein the sort channel and input channels are formed byetching trenches into the surface of the substrate, and bonding a flatplate over the substrate and the trenches; and wherein the z-focuschannel is formed with a radius of curvature of at least about 100microns and less than about 500 microns.